1. Field of the Invention
The present invention relates to a method for in vitro phosphorylation of TRAP (target of RNA III-activating protein) derived from Staphylococcus aureus and a method for screening inhibitors against the TRAP phosphorylation.
2. Description of the Related Art
Staphylococcus aureus is a Gram-positive bacterium causing food poisonings and inflammatory diseases such as skin suppuration, tympanitis and cystitis, frequently found in the nature. Staphylococcus aureus also provokes various diseases in humans and animals, because it expresses and secretes hemolytic toxins, endotoxins, exotoxins, proteases, and the like [Lowy F. D., Staphylococcus aureus infections. 1998 N. Engl. J. Med., 339:520-532]. In prior arts, it is reported that the toxin expression of Staphylococcus aureus is regulated by a transcriptional expression of RNA III. Also, the transcriptional expression of RNA III is elucidated to be regulated by a quorum sensing mechanism. In turn, the quorum sensing mechanism of Staphylococcus aureus is controlled by transcription-regulating proteins such as AIP (autoinducing peptide) and Rap (RNAIII activating protein) [Balaban N. et al., Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus. 1998 Science, 280:438-440; Novick R. P., Autoinduction and signal transduction in the regulation of staphylococcal virulence. (2003 Mol. Microbiol., 48:1429-49). In detail, staphylococcal bacteria secrete the RAP protein, when reaching a certain density at a specified stage. The resulting RAP protein binds onto another staphylococcal bacterium so as to transmit a quorum sensing signal. The TRAP protein is involved in the signal transduction of quorum sensing by the RAP protein. It is reported that the phosphorylation of the TRAP protein at the 66th histidine residue is essential prior to the signal transduction [Gov Y., Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues. [2004 J. Biol. Chem., 279:14665-72]. However, it is unknown yet how the TRAP phosphorylation is induced by the signal transduction of the RAP protein.
In addition, the signal transduction pathway for the quorum sensing is clarified to play a very important role during a staphylococcal infection. Actually, the expression of staphylococcal toxins and the biofilm formation are obstructed completely when the signal transduction is inhibited (Balaban N. et al., Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating protein [200. J. Biol. Chem., 276:2658-2667]). Recently, it is reported that the Rap, an initial protein of the quorum sensing signal reduces the staphylococcal infection of mice remarkably, when being treated with a specific antibody against the RAP protein or blocked with a RNAIII inhibiting peptide (RIP), a RAP mediated quorum sensing inhibitor [Balaban N. et al., Treatment of Staphylococcus aureus biofilm infection by quorum sensing inhibitor RIP. 2007 Antimicrobe Agents Chemother., 51:2226-2229]. It is also disclosed that the production of staphylococcal toxins decreases greatly and the bacterial infection decreases, when staphylococcal bacteria are mutated unable to express the TRAP or when its amino acid resides are substituted at a certain site so as not to be phosphorylated [Gov Y., Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues. 2004 J. Biol. Chem., 279:14665-72]. As a consequence, the Rap and the TRAP proteins of Staphylococcus are very important in initiating the host infection via the signal transduction. Especially, the TRAP phosphorylation is essential in determining the staphylococcal infection. Therefore, it is expected that inhibitors against the TRAP phosphorylation can be developed into a novel antibiotic to prevent the infection of Staphylococcus and other bacteria having similar proteins to the TRAP.
In order to identify the importance of the TRAP phosphorylation, Balaban et al. have phosphorylated the TRAP after culturing staphylococcal bacteria [Balaban N. et al., Regulation of Staphylococcus aureus pathogenesis via target of RNA III-activating protein. 2001, J. Biol. Chem., 276:2658-2667; Gov Y. Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues. 2004, J. Biol. Chem., 279:14665-72]. This technique is illustrated in U.S. Pat. No. 6,747,129. In the patent, the amino acids sequence of the TRAP and the method for TRAP phosphorylation using cultured staphylococcal bacteria by sending a quorum sensing signal via RAP protein are disclosed. In addition, Balaban et al. have reported in U.S. Pat. No. 6,291,431 that the RIP peptide can block the staphylococcal infection effectively, when it is used to inhibit the TRAP phosphorylation. This result is illustrated in a recently published paper [Cirioni et al., RNAIII-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of central venous catheter-associated Staphylococcus aureus infections. 2006, J. Infect. Dis., 193(2): 180-186]. Besides, Balaban et al. have confirmed that the RIP peptide can efficiently improve a germicidal activity, when being administered with an antibiotic, because it increases the drug sensitivity of staphylococcal bacteria [Balaban et al., RNAIII-inhibiting peptide inhibits in vivo biofilm formation by drug resistance Staphylococcus aureus. 2003, Antimicrob. Agents Chemother., 47(6): 1979-1983; Balaban et. al, Treatment of Staphylococcus aureus biofilm infection by the quorum sensing inhibitor RIP. 2007, Antimicrob. Agents Chemother., 51(6): 2226-2229]. As a consequence, it is clarified that the TRAP should be an effective target protein of staphylococcal bacteria in order to develop new antibiotics.
The TRAP is known to be phosphorylated within cells, when a quorum sensing signal is transmitted via the RAP protein. But the mechanism of TRAP phosphorylation has not been fully disclosed yet in patent documents or published papers. It is just conjectured that the Rap signal may be transferred from outside of a cell to inside through an unknown membrane protein and then, accepted by a certain protein kinase so as to phosphorylate the TRAP.
Presently, in order to measure the TRAP phosphorylation, microbes are cultured with media containing radioactive isotope-labelled phosphates. Then, a small amount of the TRAP protein is monitored in order to examine whether it binds with radioactive phosphates. This procedure is disadvantageous because it requires a lot of time and cost. Therefore, it seems that a method for in vitro phosphorylating the TRAP is more effective and convenient than the above method using radioisotopes, in order to select inhibitors against the TRAP. Unfortunately, such a technique of in vitro phosphorylation has not disclosed yet, because the TRAP protein is not similar to any other known proteins in respect of the structure and function.